Chronic viral hepatitis is a very common disease in the world. It is estimated that approximately 5% of the world population and about 10% of the population in Korea suffer from chronic viral hepatitis. In addition, chronic viral hepatitis arouses medical and social interest since the disease adversely impacts the life, especially the quality and productive aspects of life. Chronic hepatitis generally progresses to liver cirrhosis and possibly primary hepatoma which leads to death. Chronic hepatitis viral infection is the major cause of hepatoma and is considered a highly morbid disease. Therefore, the development of an effective antiviral agent is urgently needed. At present, reliable antiviral therapy for chronic viral hepatitis has yet to be developed. Although some agents have been proven partially effective in the treatment of chronic viral hepatitis, they have many disadvantages, including adverse side effects, which remain to be solved.
As one of the anti-viral agents commonly used in the past adenosine arabinoside monophosphate (Ara-AMP) is a purine-based preparation which exerts strong antiviral activity by inhibiting the activity of DNA polymerase. Early in the 1980's it was reported that Ara-AMP has a therapeutic response of 10 to 17% in the treatment of chronic hepatitis B. However, in 1987 Garcia et al. (Garcia G., Smith C. L., Weissberg J. I., et al.: Adenine arabinoside monophosphate in combination with human leukocyte interferon in the treatment of chronic hepatitis B: A randomized, double-blind, placebo-controlled trial. Ann Intern Med 107:278-285, 1987) reported that the Ara-AMP treatment group did not show any significantly greater effect as compared to the control group. Further, the administration of Ara-AMP sometimes develops very serious adverse effects, such as bone marrow suppression and irreversible neuromuscular pain. Accordingly, Ara-AMP is no longer used in the treatment of chronic viral hepatitis B.
It is generally known that interferon produced by animal cells, i.e. naturally occurring, is effective in the treatment of chronic vital hepatitis B. Natural interferon is a glycoprotein, i.e. a conjugated protein in which the nonprotein group is a carbohydrate of a low molecular weight. It was disclosed as a substance having antiviral activity in 1957. Numerous studies on its action, effect, mechanism of action, method for separation into a purified state, mass production, and the like, have been done in both the industrial and academic fields because of the interest in interferon as an antiviral agent and an anticancer agent. At present, the following kinds of interferon are known:
1) Alpha-interferon, which is induced and produced by vital infection of the leucocytes, has antiviral activity and a natural killer cell-activating effect; PA1 2 ) Beta-interferon, which is induced and produced by vital infection, especially double-strand RNA virus infection, of the fibroblasts, and has antiviral activity; and PA1 3) Gamma-interferon, which is induced and produced by immunological stimulation of lymphocytes with mitogen or antigen, and has an immunemodulating activity. PA1 P is a peptide residue of a human serum glycoprotein; PA1 S is a sugar residue of a human serum glycoprotein; PA1 Gal is a galactose residue; PA1 GA is a glutaraldehyde residue; PA1 x is an integer equal to or greater than 1; and PA1 INF is interferon and preferably recombinant interferon. PA1 P is a peptide residue of a human serum glycoprotein; PA1 S is a sugar residue of a human serum glycoprotein; PA1 Gal is a galactose residue of human serum glycoprotein; PA1 GA is a glutaraldehyde residue; PA1 x is an integer equal to or greater than 1; and PA1 INF is interferon,
Gamma-interferon is reported to have superior anticancer and antiviral activity as compared to the alpha and beta interferons.
Today genetic recombination methods produce recombinant interferon on an industrial scale such that it is widely used for its antiviral and immunomodulator activities.
The therapeutic effect of recombinant interferon for chronic hepatitis B has been reported as 30 to 40% in the West where the prevalence of chronic hepatitis B is relatively low. However, when compared with the spontaneous improvement of 15 to 25% in the untreated group, it is considered that the recombinant interferon is clinically effective in only about 10 to 15% of the chronic hepatitis B patients.
It is estimated that among the world population the hepatitis B surface antigen carrier is approximately 300 million people, of which the Asian population accounts for 80%. When those Asians are treated with recombinant interferon, the remission rate is merely 10 to 15% which is similar to the yearly spontaneous clearance rate of 16 to 17% of hepatitis e antigen. Therefore, a beneficial effect of recombinant interferon may not be recognized in the Asian population, including the Korean population.
It is known that although interferon can remove or reduce the presence of hepatitis B virus(HBV) in serum of the patient with chronic hepatitis B, HBV DNA may remain in the liver cells of some chronic hepatitis patients. Accordingly, a high relapse rate is expected when the treatment with interferon is discontinued.
Meanwhile, it has been reported that although there are minor differences in the dosage and in the treatment period, the use of recombinant interferon shows a therapeutic effect of 28 to 71% for chronic C-type hepatitis when the decrease of serum alanine aminotransferase value, which is obtained from a biochemical liver function test, is used as the criterion for judging the clinical therapeutic effect. However, since approximately half of the patients have a clinical relapse of hepatitis after treatment with recombinant interferon, it is considered that the therapeutic effect of recombinant interferon for C-type hepatitis is only transient. Accordingly, presently commercially available recombinant interferon provides only a transient effect in some B-type and C-type chronic hepatitis patients.
Recombinant interferon, which shows a transient effect in some B and C-type chronic hepatitis patients, unlike natural occurring interferon, is a polypeptide interferon having no sugar groups. Since recombinant interferon is excreted through the kidney into the urine immediately after administration, it has been noted that the uptake rate of recombinant interferon into the liver cells is lower than that of the natural interferon and, therefore, the effect of recombinant interferon is very limited. In practice, it has been determined that the clinical effect of recombinant interferon for B-and C-type chronic hepatitis is only transient in some patients and that both the rate of treatment failure and rate of relapse after cessation of treatment are significantly high. In addition, in order to enhance the therapeutic effect, a large amount of interferon should be administered. However, the use of a large dose is inevitably accompanied by adverse effects such as pyrexia, myalgia, arthralgia and bone marrow suppression. As discussed above, since the prior art recombinant interferon fails to show high efficacy as a therapeutic agent for chronic hepatitis, the development of a novel agent for the treatment of chronic hepatitis with a high therapeutic effect and minimal adverse effects is urgently needed.
The reason why the effect of interferon is transient is that although interferon can reduce virus DNA level in serum, it does not effectively act on viruses present in liver cells. This allows the hepatitis virus to continuously replicate in the liver cells. Therefore, the patients may suffer a recurrence of hepatitis after cessation of treatment with recombinant interferon.
Therefore, the most ideal method of treatment envisioned is drug-target therapy wherein the most effective antiviral agent is introduced directly into the target liver cells.
Methods where the antiviral agent is administered via a receptor specifically present only in the liver cells to inhibit the replication of viruses present in the liver cells have been attempted in the prior art. Fiume et al. (Biochem. Pharm. 35: 967, 1986) has synthesized L-SA-ara-AMP by combining an antiviral agent, 9-.beta.-D-arabino-furanosyl-adenyl-5'-monophosphate (ara-AMP), with lactosaminated serum albumin (L-SA), which is a novel protein synthesized by conjugation of serum albumin with lactose. It was learned that L-SA-ara-AMP can be specifically absorbed to a high level in the liver cells. However, L-SA-ara-AMP has not had a great effect on the treatment of hepatitis since the method for preparing lactosaminated serum albumin is very complicated and requires the use of albumin which is expensive. In addition, L-SA-ara-AMP is prepared by using a synthetic glycoprotein, which is not naturally present in the human body, and by combining ara-AMP, which is identified as being ineffective for the treatment of chronic hepatitis B.
All plasma proteins, except for albumin, present in blood are a glycoprotein containing a carbohydrate chain which terminates with a sialic acid residue. These plasma proteins are cleared from the circulating bloodstream through a series of processes via cleavage of the sialic acid residues with various enzymes. Neuraminidase exhibits its activity in mammal serum as an enzyme which removes sialic acid residues from glycoprotein (Rosenberg A., Schengrund C. L.:Sialidases. In Rosenberg A., Schengrund C. L.(Eds): Biological roles of sialic acid. New York, Plenum, 1976).
Plasma proteins, which are soluble and which circulate in the blood, lose their solubility and inherent physiological activities after a certain period of time. The mechanism of this action is present in the liver. It is known that the removal of glycoproteins is a function of the liver in view of the fact that when patients are suffering from hepatic cirrhosis or hepatitis and thus have a disorder in the mechanism for removing glycoproteins from the liver cells, the circulating asialoglycoproteins, which are not detected in the normal serum, are present in the serum of these patients. In practice, when desialylated ceruloplasmin is injected into a rabbit, the half life of desialylated ceruloplasmin is 2 minutes which is considerably shorter than 55 hours of the natural ceruloplasmin. This phenomenon is commonly demonstrated in the case of other plasma proteins. Specifically, by removing the sialyl group from the terminal end of the glycoprotein, the galactose group is exposed and recognized by a receptor present in the liver cell membrane resulting in its rapid movement into the liver cell via receptor-mediated endocytosis (RME) and in its disappearance from the circulating bloodstream.
In the 1960's, Morell and Ashwell et al. determined that when a sialyl group of ceruloplasmin is removed by neuraminidase, this plasma protein rapidly disappears from serum. They disclosed that this phenomenon is due to the uptake by the ASGP receptor present in liver cells (J. Biol. Chem., 243:155, 1968). Thereafter, it was reported that the ASGP receptor is present only in liver cells (Adv. Enzymol., 41: 99, 1974). Such specific uptake by liver cells has been identified from the fact that when asialoceruloplasmin or asialoorosomucoid, which is experimentally labelled with tritium, is injected into the living body, the isotope is selectively detected only in liver cells (Scheinberg I. H., Morell A. G., Stockert R. J.: Hepatic removal of circulating proteins. Davidson C. S., ed. Problems in Liver Diseases. pp 279-285, New York, Stratton Company, 1979). In addition, it was also disclosed that this receptor specifically recognizes and absorbs glycoproteins having D-galactose or N-acetylgalactosamin as the terminal sugar group (Ann. Rev. Biochem., 51; 531, 1982). The cell membrane of liver cells comprises a cell structure which combines with asialoglycoprotein terminated with galactose. This cell structure was first named hepato-binding protein (HBP) but is presently called asialoglycoprotein receptor. Further, it has been observed that among various desialylated glycoproteins, the desialylated alpha(1)-acid glycoprotein, asialoorosomucoid, most rapidly disappears from the serum after injection. Therefore, it has been determined that asialo-alpha(1)-acid glycoprotein is both specifically and well taken up by liver cells (FIG. 1) (J. Biol. Chem., 245; 4397, 1970).
Natural interferon which has remarkable antiviral activity is also a glycoprotein containing sialic acid at the terminal end of a carbohydrate chain and it is rapidly cleared from the bloodstream after intravenous injection. Asialo-interferon terminated with galactose, which is prepared by treating naturally occurring interferon with an enzyme to remove the terminal sialyl group, is rapidly cleared from the blood-stream in comparison to naturally occurring interferon, as was also demonstrated in the cases of other glycoproteins including ceruloplasmin.
However, it has been reported that the removal of over 85% of the carbohydrate moiety from the interferon molecule by treatment with glycosidase in order to retain only the polypeptide portion, does not alter the antiviral activity of interferon but reduces the uptake by liver cells which increases the half-life of interferon in the blood (Bose S., Hickman J.: Role of carbohydrate moiety in determining the survival of interferon in the circulation, J. Biol. Chem., 252: 8336, 1977; FIG. 2). Thus, it has been suggested that asialo-interferon, like other asialo-plasma glycoproteins, also passes through a pathway of selective uptake via the asialo-glycoprotein receptor into the liver cells which accounts for its disappearance from the circulating bloodstream.
In 1980, Weissmann et al. prepared a recombinant interferon by manipulation of the INF-.alpha..sub.2b gene of the human leukocyte into E. coli. Since recombinant interferon consists only of 165 amino acids and is without carbohydrate chains, the uptake of recombinant interferon into the liver cells is very poor, like the polypeptide interferon formed by treatment of the naturally occurring interferon with glycosidase.
Therefore, it is an object of this invention to target recombinant interferon to the asialoglycoprotein(ASGP) receptors present specifically in the liver cells to enhance the antiviral activity of the recombinant interferon against the hepatitis viruses which almost exclusively replicate in the liver cells.
It is a further object of this invention to provide an interferon-conjugate which can be administered to mammals, including humans, who show the clinical, serological and molecular biological evidence of hepatitis, to treat hepatitis viral infection, including hepatitis types B, C and D.
It is a further object of this invention to provide a method for treating viral hepatitis by administering to a patient in need, an amount of the conjugated interferon of formula (1), as set forth below, sufficient to inhibit the replication of hepatitis virus present in the liver cells of the patient. It is a further object of this invention to provide a composition for treating viral hepatitis comprising an amount of a conjugated interferon of formula (1), set forth below, effective to inhibit the replication of hepatitis virus present in the liver cells and a pharmaceutically acceptable carrier, adjuvant or excipient therefor.
It is a further object of this invention to provide a method of preparing the novel conjugated antiviral agents by combining asialoglycoprotein(ASGP) with recombinant interferon (INF) resulting in an interferon-conjugate which when parenterally administered to a patient, becomes bound to the ASGP receptor specifically present in the liver cells.
It is an advantage of this invention to provide an interferon-conjugate which reduces the manifestation of adverse reactions relative to the manifestation of adverse reactions of recombinant interferon alone.
The foregoing has outlined some of the more pertinent objects of the present invention. These objects should be construed to be merely illustrative of some of the more pertinent features and applications of the invention. Many other beneficial results can be obtained by applying the disclosed invention in a different manner or modifying the invention within the scope of the disclosure. Accordingly, other objects and a more thorough understanding of the invention may be had by referring to the summary of the invention and the detailed description describing the preferred embodiment in addition to the scope of the invention defined by the claims taken in conjunction with the accompanying drawings.